The present invention relates generally to oligonucleotide primer extension methods for identifying a single nucleotide in a nucleic acid sample. Methods of the invention are useful for disease diagnosis by detecting and identifying the presence of genetic mutations or disease-causing microorganisms in biological samples.
The knowledge of molecular defects causative of diseases, such as inherited disorders and cancer, is increasing rapidly. Inherited diseases thought to be caused by genetic mutations include sickle cell anemia, xcex1- and xcex2-thalassemias, phenylketonuria, hemophilia, xcex1i-anti-trypsin deficiency, and cystic fibrosis. Sickle cell anemia, for example, is reported to result from homozygosity resulting from a single base pair substitution in the sixth codon of the xcex2-globin gene. Antonarakis, New England J. Med., 320: 153-163 (1989). Mutations in the insulin receptor gene and in the insulin-responsive glucose transporter gene have been detected in insulin-resistant diabetes. Krook et al., Human Molecular Genetics, 1: 391-396 (1992).
Cancer has been associated with genetic mutations in a number of oncogenes and tumor suppressor genes. Duffy, Clin. Chem., 41: 1410-1413 (1993). For example, point mutations in the ras genes have been shown to convert those genes into transforming oncogenes. Bos et al., Nature, 315: 726-730. Mutations and the loss of heterozygosity at the p53 tumor suppressor locus have been correlated with various types of cancer. Ridanpaa et al., Path. Res. Pract., 191: 399-402 (1995); Hollstein et al., Science, 253: 49-53 (1991). In addition, the loss or other mutation of the apc and dcc tumor suppressor genes has also been associated with tumor development. Blum, Europ. J. Cancer, 31A: 1369-1372 (1995). Those mutations can serve as markers for early stages of disease and for predisposition thereto. Early diagnosis is not only important for successful treatment, but can also lead to prevention or treatment before chronic symptoms occur.
Colorectal cancer is an example of a disease that is highly curable if detected early. With early detection, colon cancer may be effectively treated by, for example, surgical removal of the cancerous tissue. Surgical removal of early-stage colon cancer is usually successful because colon cancer begins in cells of the colonic epithelium and is isolated from the general circulation during its early stages. Thus, detection of early mutations in colorectal cells would greatly increase survival rate. Current methods for detection of colorectal cancer focus on extracellular indicia of the presence of cancer, such as the presence of fecal occult blood or carcinoembryonic antigen circulating in serum. Such extracellular indicia typically occurs only after the cancer has become invasive. At that point, colorectal cancer is very difficult to treat.
Methods have been devised to detect the presence of mutations within disease-associated genes. One such method is to compare the complete nucleotide sequence of a sample genomic region with the corresponding wild-type region. See, e.g., Engelke et al., Proc. Natl. Acad. Sci, U.S.A., 85: 544-548 (1988); Wong et al., Nature, 330: 384-386 (1988). However, such methods are costly, time consuming, and require the analysis of multiple clones of the targeted gene for unambiguous detection of low-frequency mutations. As such, it is not practical to use extensive sequencing for large-scale screening of genetic mutations.
A variety of detection methods have been developed which exploit sequence variation in DNA using enzymatic and chemical cleavage techniques. A commonly-used screen for DNA polymorphisms consists of digesting DNA with restriction endonucleases and analyzing the resulting fragments by means of southern blots, as reported by Botstein et al., Am. J. Hum. Genet., 32: 314-331 (1980) and White et al., Sci. Am., 258: 40-48 (1988). Mutations that affect the recognition sequence of the endonuclease will preclude enzymatic cleavage at that site, thereby altering the cleavage pattern of the DNA. Sequences are compared by looking for differences in restriction fragment lengths. A problem with this method (known as restriction fragment length polymorphism mapping or RFLP mapping) is its inability to detect mutations that do not affect cleavage with a restriction endonuclease. One study reported that only 0.7% of the mutational variants estimated to be present in a 40,000 base pair region of human DNA were detected using RFLP analysis. Jeffreys, Cell, 18: 1-18 (1979).
Single base mutations have been detected by differential hybridization techniques using allele-specific oligonucleotide (ASO) probes. Saiki et al., Proc. Natl. Acad. Sci. USA, 86: 6230-6234 (1989). Mutations are identified on the basis of the higher thermal stability of the perfectly-matched probes as compared to mismatched probes. Disadvantages of this approach for mutation analysis include: (1) the requirement for optimization of hybridization for each probe, and (2) the nature of the mismatch and the local sequence impose limitations on the degree of discrimination of the probes. In practice, tests based only on parameters of nucleic acid hybridization function poorly when the sequence complexity of the test sample is high (e.g., in a heterogeneous biological sample). This is partly due to the small thermodynamic differences in hybrid stability generated by single nucleotide changes. Therefore, nucleic acid hybridization is generally combined with some other selection or enrichment procedure for analytical and diagnostic purposes.
In enzyme-mediated ligation methods, a mutation is interrogated by two oligonucleotides capable of annealing immediately adjacent to each other on a target DNA or RNA molecule, one of the oligonucleotides having its 3xe2x80x2 end complementary to the point mutation. Adjacent oligonucleotide sequences are only covalently attached when both oligonucleotides are correctly base-paired. Thus, the presence of a point mutation is indicated by the ligation of the two adjacent oligonucleotides. Grossman et al., Nucleic Acid Research, 22: 4527-4534 (1994). However, the usefulness of this method for detection is compromised by high backgrounds which arise from tolerance of certain nucleotide mismatches or from non-template directed ligation reactions. Barringer et al., Gene, 89: 117-122 (1990).
A number of detection methods have been developed which are based on a template-dependent, primer extension reaction. These methods fall essentially into two categories: (1) methods using primers which span the region to be interrogated for the mutation, and (2) methods using primers which hybridizes proximally and upstream of the region to be interrogated for the mutation.
In the first category, Caskey and Gibbs [U.S. Pat. No. 5,578,458] report a method wherein single base mutations in target nucleic acids are detected by competitive oligonucleotide priming under hybridization conditions that favor the binding of the perfectly-matched primer as compared to one with a mismatch. Vary and Diamond [U.S. Pat. No. 4,851,331] described a similar method wherein the 3xe2x80x2 terminal nucleotide of the primer corresponds to the variant nucleotide of interest. Since mismatching of the primer and the template at the 3xe2x80x2 terminal nucleotide of the primer inhibits elongation, significant differences in the amount of incorporation of a tracer nucleotide result under normal primer extension conditions.
It has long been known that primer-dependent DNA polymerases have, in general, a low replication error rate. This feature is essential for the prevention of genetic mistakes which would have detrimental effects on progeny. Methods in a second category exploit the high fidelity inherent in this enzymological reaction. Detection of mutations is based on primer extension and incorporation of detectable, chain-terminating nucleoside triphosphates. The high fidelity of DNA polymerases ensures specific incorporation of the correct base labeled with a reporter molecule. Such single nucleotide primer-guided extension assays have been used to detect aspartylglucosaminuria, hemophilia B, and cystic fibrosis; and for quantifying point mutations associated with Leber Hereditary Optic Neuropathy (LHON). See. e.g., Kuppuswamy et al., Proc. Natl. Acad. Sci. USA, 88: 1143-1147 (1991); Syvanen et al., Genomics, 8: 684-692 (1990); Juvonen et al., Human Genetics, 93: 16-20 (1994); Ikonen et al., PCR Meth. Applications, 1: 234-240 (1992); Ikonen et al., Proc. Natl. Acad. Sci. USA, 88: 11222-11226 (1991); Nikiforov et al., Nucleic Acids Research, 22: 4167-4175 (1994). An alternative primer extension method involving the addition of several nucleotides prior to the chain terminating nucleotide has also been proposed in order to enhance resolution of the extended primers based on their molecular weights. See e.g., Fahy et al., WO/96/30545 (1996).
Strategies based on primer extension require considerable optimization to ensure that only the perfectly annealed oligonucleotide functions as a primer for the extension reaction. The advantage conferred by the high fidelity of the polymerases can be compromised by the tolerance of nucleotide mismatches in the hybridization of the primer to the template. Any xe2x80x9cfalsexe2x80x9d priming will be difficult to distinguish from a true positive signal.
The selectivity and sensitivity of an oligonucleotide primer extension assay are related to the length of the oligonucleotide primer, and to the reaction conditions. In general, primer lengths and reaction conditions that favor high selectivity result in low sensitivity. Conversely, primer lengths and reaction conditions that favor high sensitivity result in low selectivity.
Under typical reaction conditions, short primers (i.e., less than about a 15-mer) exhibit transient, unstable hybridization. Therefore, the sensitivity of a primer extension assay is low when a short primer is used, because a transient, unstable oligonucleotide hybrid does not readily prime the extension reaction, resulting in a low yield of extended oligonucleotide. Moreover, in a complex heterogeneous biological sample, short primers exhibit non-specific binding to a wide variety of perfectly-matched complementary sequences. Thus, because of their low stability and high non-specific binding, short primers are not very useful for reliable identification of a mutation at a known location. Therefore, detection methods based on primer extension assays use oligonucleotide primers ranging in length from 15-mer to 25-mer. See e.g., PCT Patent Publications WO 91/13075; WO 92/15712; and WO 96/30545. Lengthening the probe to increase stability, however, has the effect of diminishing selectivity. A single base mismatch usually has less effect on the binding efficiency of a longer oligonucleotide primer than it does on that of a shorter primer, because of the relatively smaller thermodynamic difference between a mismatched primer and a perfectly matched primer. This higher tolerance of nucleotide mismatches in the hybridization of the longer primer to the template can result in higher levels of non-specific xe2x80x9cfalsexe2x80x9d priming in complex heterogeneous biological samples.
The reaction conditions of a primer extension reaction can be optimized to reduce xe2x80x9cfalsexe2x80x9d priming due to a mismatched oligonucleotide. However, optimization is labor intensive and expensive, and often results in lower sensitivity due to a reduced yield of extended primer. Moreover, since considerable optimization is required to ensure that only the perfectly annealed oligonucleotide functions as a primer for the extension reaction, only limited multiplexing of the primer extension assays is possible. Krook et al., supra report that multiplexing can be achieved by using primers of different lengths and by monitoring the wild-type and mutant nucleotide at each mutation site in two separate single nucleotide incorporation reactions. However, given that the selectivity and stability of the oligonucleotide primer extension assay is determined by the length of the oligonucleotide primer and the reaction conditions, the number of primers that can be tested simultaneously in a given reaction mixture is very limited.
Methods in the art reduce the possibility of false priming by decreasing the sequence complexity of the test sample. Thus, genomic DNA is isolated from the biological sample and/or amplified with PCR using primers which flank the region to be interrogated. The primer extension analysis is then conducted on the purified PCR products. See PCT Patent Publications WO 91/13075; WO 92/15712; and WO 96/30545. However, these methods are time consuming and expensive, because they involve additional steps of sample processing. Furthermore, these methods are not adapted for multiple primer extension reactions in a single sample.
Therefore, there is a need in the art for a selective and sensitive nucleic acid detection method, and for reliable large-scale screening methods for a large number of genomic mutations in heterogeneous biological samples. Such methods are provided herein.
The invention provides methods of mutation detection having high sensitivity and high selectivity. In a general embodiment, the invention comprises a single base extension reaction that is repeated at least once. Methods of the invention are useful to detect and identify genetic mutations or the presence of disease-causing microorganisms in an heterogeneous biological sample.
Methods of the invention comprise conducting multiple cycles of a single-base extension reaction, thereby increasing the sensitivity of the primer extension assay without compromising the selectivity. In a preferred embodiment, methods of the invention comprise between 2 and 100 cycles of primer extension. More preferably, between 10 and 50 cycles are performed. Most preferably, approximately 30 cycles are performed.
In a preferred embodiment, an excess of primer is used, to ensure that additional extended primer products are produced in each extension cycle. The oligonucleotide primer length is preferably between about 10 to about 100 nucleotides, more preferably between about 15 and about 35 nucleotides, and most preferably about 25 nucleotides.
In a preferred embodiment, each extension reaction includes conditions that promote hybridization of the primer only to nucleic acids with a perfect complementary sequence (i.e. mismatched base pairs are not tolerated). In one embodiment, the hybridization is performed at about the Tm for the primer in the assay. In a more preferred embodiment, the hybridization is performed above the Tm for the primer.
In one embodiment, a hybridized oligonucleotide primer is extended with a labeled terminal nucleotide. Labeled ddNTPs or dNTPs preferably comprise a xe2x80x9cdetection moietyxe2x80x9d which facilitates detection of the extended primer. Detection moieties are selected from the group consisting of fluorescent, luminescent or radioactive labels, enzymes, haptens, and other chemical tags such as biotin which allow for easy detection of labeled extension products by, for example, spectrophotometric methods.
In a preferred embodiment, a further cycle of primer extension is started by denaturing the hybridized and extended primer, annealing nonextended primer, and extending the newly hybridized primer. The presence of excess primer in the reaction promotes annealing of nonextended primer in each cycle of the reaction.
In a further embodiment, methods of the invention comprise conducting at least two cycles of a single-base extension reaction using segmented primers. Methods of the invention comprise hybridizing two probes adjacent to a site of suspected mutation, wherein neither probe alone is capable of being a primer for template-dependent extension, but when the probes hybridize adjacent to each other, they are capable of priming extension. In a preferred embodiment, methods of the invention comprise hybridizing to a target nucleic acid a probe having a length from about 5 bases to about 10 bases, wherein the probe hybridizes immediately upstream of a suspected mutation. Methods of the invention further comprise hybridizing a second probe upstream of the first probe, the second probe having a length from about 15 to about 100 nucleotides and having a 3xe2x80x2 non-extendible nucleotide. The second probe is substantially contiguous with the first probe. Preferably, substantially contiguous probes are between 0 and about 1 nucleotide apart. A linker is preferably used where the first and second probes are separated by two or more nucleotides, provided the linker does not interfere with the nucleic acid extension reaction. Such linkers are known in the art and include, for example, peptide nucleic acids, DNA binding proteins, and ligation.
In an alternative embodiment, segmented primers comprise a series of first oligonucleotide probes. No member of the series of the first probes is capable of being a primer for nucleic acid polymerization unless every member of said series hybridize simultaneously to substantially contiguous portions of the target nucleic acid, thereby forming a contiguous primer. In one embodiment, the segmented primers comprise three 8-mer first probes. In another embodiment, the segmented primers comprise four 6-mer first probes.
In each cycle of the extension assay, an extension reaction adds nucleotides to the segmented primer resulting from co-hybridization of the above-described probes in a template-dependent manner. In a preferred embodiment, first probes hybridized to a target nucleic acid are extended with a labeled terminal nucleotide whereas first probes hybridized to a wild-type or non-target nucleic acid are extended with an unlabeled terminal nucleotide. Labeled ddNTPs or dNTPs preferably comprise a xe2x80x9cdetection moietyxe2x80x9d which facilitates detection of the short probes that have been extended with a labeled terminal nucleotide. Detection moieties are selected from the group consisting of fluorescent, luminescent or radioactive labels, enzymes, haptens, and other chemical tags such as biotin which allow for easy detection of labeled extension products by, for example, spectrophotometric methods.
In a preferred embodiment, several cycles of extension reactions are conducted in order to amplify the assay signal. Extension reactions are conducted in the presence of an excess of first and second probes, labeled dNTPs or ddNTPs, and heat-stable polymerase. Once an extension reaction is completed, the first and second probes bound to target nucleic acids are dissociated by heating the reaction mixture above the melting temperature of the hybrids. The reaction mixture is then cooled below the melting temperature of the hybrids and first and second probes are permitted to associate with target nucleic acids for another extension reaction. In a preferred embodiment, 10 to 50 cycles of extension reactions are conducted. In a most preferred embodiment, 30 cycles of extension reactions are conducted.
Methods disclosed herein may be used to detect single nucleotide polymorphisms (SNPs), mutations such as insertions, deletions, and substitutions. Nucleic acid samples that can be screened with the methods of the present invention include human nucleic acid samples. A primer (or segmented primer) is designed so that the 3xe2x80x2 end of the hybridized primer is immediately upstream of the position that is complementary to the nucleotide position being assayed. The nucleotide position being assayed is identified as the nucleotide that is complementary to the nucleotide incorporated in the single-base primer extension reaction. For example, if a G is incorporated in the reaction, a C is present at the complementary position on the nucleic acid in the biological sample. In a preferred embodiment, a primer extension reaction is performed in the presence of four nucleotides, preferably chain terminating nucleotides, for example the dideoxynucleotides ddATP, ddCTP, ddGTP, and ddTTP. In a more preferred embodiment, the nucleotides are detectably labeled, preferably differentially labeled. In alternative embodiments, the extension reaction is performed in the presence of one, two, or three different nucleotides. If the biological sample is heterogeneous at the nucleotide position being assayed, the complementary nucleotides (if they are included in the primer extension reaction) will be incorporated in the primer extension assay.
Methods disclosed herein may be used to detect mutations associated with diseases such as cancer. Additionally, methods of the invention may be used to detect a deletion or a base substitution mutation causative of a metabolic error, such as complete or partial loss of enzyme activity.
In another embodiment, the specific nucleic acid sequence comprises a portion of a particular gene or genetic locus in the patient""s genomic nucleic acid known to be involved in a pathological condition or syndrome. Non-limiting examples include cystic fibrosis, Tay-Sachs disease, sickle-cell anemia, xcex2-thalassemia, and Gaucher""s disease.
In yet another embodiment, the specific nucleic acid sequence comprises part of a particular gene or genetic locus that may not be known to be linked to a particular disease, but in which polymorphism is known or suspected.
In yet another embodiment, the specific nucleic acid sequence comprises part of a foreign genetic sequence e.g. the genome of an invading microorganism. Non-limited examples include bacteria and their phages, viruses, fungi, protozoa, and the like. The present methods are particularly applicable when it is desired to distinguish between different variants or strains of a microorganism in order to choose appropriate therapeutic interventions.
Genomic nucleic acid samples are isolated from a biological sample. Once isolated, the nucleic acids may be employed in the present invention without further manipulation. Alternatively, one or more specific regions present in the nucleic acids may be amplified by, for example, PCR. Amplification at this step provides the advantage of increasing the concentration of specific nucleic acid sequences within the target nucleic acid sequence population. In another embodiment, genomic nucleic acids are fragmented before further analysis.
In one embodiment, the nucleic acids are bound to a solid-phase support. This allows the simultaneous processing and screening of a large number of samples. Non-limiting examples of supports suitable for use in the present invention include nitrocellulose or nylon filters, glass beads, magnetic beads coated with agents for affinity capture, treated or untreated microtiter plates, and the like. In a preferred embodiment, the support is a microtiter dish, having a multiplicity of wells. The use of such a support allows the simultaneous determination of a large number of samples and controls, and thus facilitates the analysis. Moreover, automated systems can be used to provide reagents to such microtiter dishes. In an alternative embodiment, methods of the invention are conducted in an aqueous phase.
In one embodiment of the invention, the extended primers or probes are enumerated. The primers or probes are preferably extended with a nucleotide labeled with an impedence bead, and the number of impedence beads is counted (using for example a Coulter counter). The number of labeled primers is then determined from the number of impedence beads. The label is more preferably a radioactive isotope, and the amount of radioactive decay associated with the labeled primer or probe is determined. The number of labeled primers or probes is calculated from the amount of radioactive decay. The numbers of extended primers or probes are useful for a statistical analysis of the cycled extension reaction.
Finally, methods of the invention further comprise isolating and sequencing the extended primers or first probes. Primers or first probes preferably comprise a xe2x80x9cseparation moietyxe2x80x9d that facilitates their isolation. Non-limiting examples of separation moieties include hapten, biotin, and digoxigenin. In a preferred embodiment, primers or first probes comprising a separation moiety are immobilized to a solid support having affinity for the separation moiety (e.g., coated with anti-hapten, avidin, streptavidin, or anti-digoxigenin). The solid support is selected from the group consisting of glass, plastic, and paper. The support is fashioned as a column, bead, dipstick, or test tube. In a preferred embodiment, the separation moiety is incorporated in the labeled ddNTPs or dNTPs and only first probes extended with a labeled ddNTP or dNTP are immobilized to the support. As such, labeled primers or first probes are isolated from unextended primers or first probes and second probes. In an alternative preferred embodiment, the separation moiety is incorporated in all the first probes, provided the separation moiety does not interfere with the first probe""s ability to hybridize with template and to be extended. By incorporating the separation moiety in the first probes, all first probes are immobilized to a solid support. First probes are isolated from second probes by one or more washing steps.
Labeled primers or first probes are then sequenced to identify a mutation or disease-causing microorganism. In one embodiment, the immobilized primers or probes are directly subjected to sequencing, using for example, chemical methods standard in the art. In other embodiments, the labeled first probes are removed from the solid support and sequencing of labeled first probes is performed in aqueous solution. The isolated first probes are contacted with a multiplicity of complementary oligonucleotides. In one embodiment, enzymatic sequencing is performed using the isolated first probes as primers and the complementary oligonucleotides as templates. In an alternative embodiment, a single base extension reaction is performed using the isolated first probes as primers and the complementary oligonucleotides as templates. The sequence of the extension product is determined by enzymatic sequencing. The sequence of the extended labeled first probes identifies the genetic mutations or the disease-causing microorganisms present in the sample.
Further aspects and advantages of the invention are apparent upon consideration of the following detailed description thereof.